Tunicamycin-induced a concentration dependent decrease in cytochrome P-450 in rat hepatocyte with a maximum decrease of 44% by 1 ug/ml tunicamycin after 3 days. It also decreased NADPH-cytochrome c reductase to a lesser extent. The effect of tunicamycin on these two systems were not evident till after 24 hr of incubation. However, the inhibitory effect of tunicamycin on protein synthesis was evident as early as 2 hr and was maximum at 24 hr. When the proteins were separated by SDS PAGE, There was a marked reduction in cytochrome P-450 isozymes as well as marked reduction in the incorporation of [35S]-methionin in these isozymes by tunicamycin. Various homologs of tunicamycin also decreased both cytochrome P-450 and protein synthesis in hepatocytes. The effect of tunicamycin on protein synthesis as well as cytochrome P-450 was due to a reduction in the synthesis of mRNA was investigated by the effect of tunicamycin on (14C)-uridine incorporation into RNA and isolation of RNA followed by translation of RNA into cytochrome P-450. Tunicamycin reduced total RNA synthesis as well as a reduction in the synthesis of mRNA coding for cytochrome P-450. Tunicamycin did not have any effect on the replication of DNA even though it inhibited DNA repair.